Histone H1 facilitates restoration of H3K27me3 during DNA replication by chromatin compaction

During cell renewal, epigenetic information needs to be precisely restored to maintain cell identity and genome integrity following DNA replication. The histone mark H3K27me3 is essential for the formation of facultative heterochromatin and the repression of developmental genes in embryonic stem cells. However, how the restoration of H3K27me3 is precisely achieved following DNA replication is still poorly understood. Here we employ ChOR-seq (Chromatin Occupancy after Replication) to monitor the dynamic re-establishment of H3K27me3 on nascent DNA during DNA replication. We find that the restoration rate of H3K27me3 is highly correlated with dense chromatin states. In addition, we reveal that the linker histone H1 facilitates the rapid post-replication restoration of H3K27me3 on repressed genes and the restoration rate of H3K27me3 on nascent DNA is greatly compromised after partial depletion of H1. Finally, our in vitro biochemical experiments demonstrate that H1 facilitates the propagation of H3K27me3 by PRC2 through compacting chromatin. Collectively, our results indicate that H1-mediated chromatin compaction facilitates the propagation and restoration of H3K27me3 after DNA replication.

In this manuscript, Liu and co-workers aim to address the role of histone H1 in restoration of K27me3 levels after replication. This is an important question and the ChORseq method is the appropriate tool to address this. The authors use ChORseq with spike-in correction to analyse restoration kinetics and conclude that repressed regions with high H3K27me3 levels restore most rapidly (consistent with findings in HeLa cells, Reveron-Gomez et al., 2018). They conclude that the restoration rate of H3K27me3 is pivotal for gene silencing, but no causative data are presented (repressed genes restore most rapidly, this does not mean that rapid restoration is required for silencing). The authors go on to show a positive correlation of the restoration kinetics to H1 occupancy, which reflects that there is more H1 at high occupancy K27me3 sites (known to restore most rapidly). The authors take advantage of H1-TKO to address the role of H1 in K27me3 post-replication restoration with the caveat that H3K27me3 levels are 2-fold reduced in this background (consistent with previous findings, Willcockson et al., 2021). The authors conclude that H1 governs H3K27me3 restoration kinetics. To support this model they show i) in vitro that H1 compaction of a chromatin array enhances PRC2 activity and K27me3 spreading (consistent with evidence on di-nucleosomes and cell-based analysis, Willcockson et al., 2021) and ii) that establishment of a K27me3 domain is less efficient in a H1 TKO background. Finally, they present a compelling model. I overall find this story compelling, mainly because the model is highly consistent with earlier studies and most probably correct. It is well documented by the Skoultchi and Zhu labs that PRC2 activity and H3K27me3 occupancy is dependent on compaction and histone H1 (conclusions of Figure 5 and Figure  3). The novelty of this manuscript would be to demonstrate the relevance of this in restoration after replication. However, the conclusions on H3K27me3 restoration are not supported by the data as there are serious issues with the ChORseq experimental setup and data analysis. Also, a number of conclusions on causality are based on correlative observations. These serious concerns must be addressed before publication of the manuscript can be recommended.

Major points
The authors perform ChORseq in a synchronization set-up where mESCs are labelled after release into S phase 2hrs. There is no analysis of EdU labelled DNA and the genomic coverage, thus it is unclear what part of the genome they are tracking. If the aim is to address H3K27me3 dynamics across the genome and identify key parameters for restoration kinetics, the full genome must be covered by EdU labelling. The authors therefore would need to repeat the ChOR-seq experiments by labelling asynchronous cells.
There is little information provided about the data analysis, but it appears that the ChORseq analysis is done without filtering for replicated regions, which is essential in a synchronization set up. By including regions that are not covered by the EdU labelling, conclusions will be based on changes in background signal (i.e, regions that have not been replicated in a high proportion of the cells or not replicated at all in the 15min EdU pulse during early/mid S).
The use of a synchronization setup is also problematic for the comparison between WT and H1 TKO cells, as it assumes that the same set of regions will be replicating during the EdU pulse in early/mid S in both cell lines. This problem would be circumvented by doing ChORseq in asynchronous cells.
The information provided on data analysis is very limited. The authors refer to the software but do not specify how they normalize the signal, how the different units are computed (for example, read density) or how peaks are defined (from ChIP, from ChORseq…). Without this information it is not possible to evaluate the conclusions based on quantitative differences, for example. They do also not specify how the different gene categories are defined; i.e. H3K27me3 peaks are generally not found on housekeeping promoters so what are they looking at?
The major conclusions of Figure 3 and Figure 5 were published previously by Fan et al., 2005 andWillcockson et al., 2021, reducing overall novelty of the work. H3K27me3 restoration kinetics was also previously shown to be fastest at sites with high K27me3 and PRC2 binding (Reveron-Gomez et al., 2018). It would be advisable to cite these publications when concluding on experiments that validate or corroborate previously findings (even if the papers are cited elsewhere in the manuscript).
Given that the K27me3 level is 2-fold reduced in the H1 TKO cells, it not clear whether the lower K27me3 or lack of H1 is responsible for the reduced restoration kinetics. An inducible system with similar baseline prior to replication would be preferable. The authors could also compare in WT cells restoration kinetics of regions with similar K27me3 levels and different H1 density.
There are conclusions on causality based on correlations mainly concerning the importance of restoration rates for gene regulation.

Minor points
It is unclear what data was produced in this manuscript and what data comes from published datasets. There are no accession numbers or references to any published data.
The authors refer to different genomic regions without defining them or providing any information on their overlap, e.g. they classify first restoration kinetics of peaks and then calculate the restoration kinetics to pre-defined promoters (ES, housekeeping, bivalent etc.). It is also not clear if these promoters have H3K27me3.
Details missing in the figures and figure legends, mostly units and data source.
Details missing in the methods, e.g there is a second thymidine addition in the schematic in Fig.1 but this is not clear from the methods.
Reviewer #2 (Remarks to the Author): Mechanisms of propagation and maintenance of epigenetic regulation across cell cycle are of significant interest, both because of fundamental importance to developmental biology, and due to emerging understanding that many human disorders are driven by misregulation of gene expression. The idea that repressive chromatin modifications are particularly dependent on the "inheritance" mechanisms during S phase has been developed in significant detail by many laboratories, including Reinberg (e.g., Reinberg andVales, Science 2018, andEscobar et al., Cell 2019) [e.g., Zhanmg et al., Plos Genet 2012] but given the complexity of the phenotypes, specific relationship between H1 incorporation and PRC2-driven H3K27 methylation must be studied further. The current study attempts to tackle this question using several experimental approaches. First, the authors study H3K27me dynamics in post-replication chromatin using ChOR-Seq. Second, they generate and characterize H1 triple-knockout cell line. Third, they perform several biochemical assays to assess the impact of H1 incorporation on PRC2 function in vitro.
While the paper is undoubtedly timely and the question under investigation is of broad interest and significance, I have several concerns about both the experimental setup and outcomes.
1. I have some concern about the ChOR-Seq setup. Replication is not uniform across the S-phasewith repressive chromatin generally contained in late-replicating domains. If the cells are indeed synchronized at the onset of S phase (as stated at line 187 of supplementary material -although Fig  S4A argues that quite a few cells are in the middle of S?), and S phase takes several hours to complete, I would expect that early-replicating domains, predominantly found in active chromatin, would be the first to incorporate EdU -and more delayed-replicating domains, located in K27merepressed (and ultimately K9me-repressed) chromatin would engage in replication much later. A recent paper by Glibert group (Zhao, Sasaki and Gilbert, Genome Biol 2020) has mapped several classes of initiation zones in mES cells, with roughly 1500 initiating early, and ~1000 initiating later in the S-phase. At the very least, I'd like to know how do these correspond to ChOR-Seq data in the current manuscript? Given that, to the naked eye, most robust changes in K27me3 happen between T0 and T2 (Figure S1B, and Figure 4A, B, D), I'd like to know more about these regions (it is somewhat counterintuitive that K27me3-rich regions are replicated within first two hours of release).
2. Perhaps even more puzzling, looking at the data in Fig S1B, I DO NOT see three distinct classes of K27me3 restoration. Even though the peaks are stated to be scaled by parental K27me3 density (or peak height?), they all look virtually identical both in their dynamics (change from T0 to T6) and original peak intensity (top to bottom in "Parental" heatmap. Given that much of the paper is based on interpretation of these data, it's critical to show that there are indeed distinct restoration clusters. Figure 1B looks like it, and perhaps some of the discrepancy is due to the scale differences (S1B is scaled -3 to 3, and 1B is scaled -1 to 1). Yet I do not see how clusters A, B and C are derived from raw data shown in S1B.
3. As overall steady-state levels of K27me3 are different between the two cell lines used in the study (see fig. 3C, D -and previously reported e.g. by Willcockson et al., Nature 2021), and pre-existing K27me3 levels determine the rate of K27me3 spreading, I am not convinced that this system allows the authors to unequivocally state that H1 loss is directly affecting the rate of spreading. We know that H1 appears to stimulate PRC2 even in di-nucleosomal substrates (first reported by Martin et al., JBC 2006) -I think the jump to compaction as mechanism is intriguing and very plausible, but is not fully supported by data. Further, comparing data in 2C-G, I am left with the impression that the greatest predictor of K27me3 restoration rate (Fig. 2C) is K27me3 read density ( Fig 2D) -not the H1e read density (Fig 2E).
4. The RNA-Seq in H1 TKO cells is a bit puzzling given almost identical number of genes going up and down. I would have expected more genes being upregulated given how dramatically H3K27me3 appears to be affected (and given what we know about H1 as a repressor). The GSEA plots are not informative as shown. Do the upregulated genes encompass sets found within cluster A? What are the downregulated genes? 5. The previous concern could be partially alleviated by a more robust distinction between recruitment and spreading. If H1 is indeed affecting the spreading dynamics, then the K27me3 peak height should remain roughly similar -but the breadth is expected to be reduced dramatically. At the very least, I'd expect a faster dropoff (such that K27me3 peaks in WT would essentially represent "obtuse angles", and in TKO would be more akin to "acute angles") from the peak center to the edges. The authors do the right experiment in Fig 5E-F, but looking at the data, I don't see a difference in K27me3 peak shape -only an overall decrease in H1 TKO background -therefore, I still can't parse out whether H1 loss causes defect in "recruitment" or "spreading" in vivo.
6. Several essential controls are missing: a. While the original H1 TKO lines generated by Skoultchi lab have been extensively characterized and show about 2-fold reduction of total H1 levels, the newly generated lines reported in the paper require additional characterization. What is the genomic landscape of CRISPR-generated lines? Did Cas9 editing create large deletions or small substitutions? How were the clones isolated (or are these nonclonal lines?) What are the levels of H1 (can be estimated by HPLC, or even by Coomassie staining, since H1 isoforms have an obvious migration pattern -this is critical since H1 genes are dosagecompensated!) b. are K27me3 levels uniform in these cells? (metastable mES cells grown in serum/LIF media have significant variability in H3 K27me3 levels -are the K27me3 "low" cells cycling faster or slower? (basically, which cell state contributes most signal to the ChOR?). I am not sure "original" TKO cells from Skoultchi lab were ever tested for K27me3 heterogeneity, and interestingly, the original report (Fan et al., Cell 2005) did not report significant changes in K27me -but it seems like an important question. Perhaps analyses similar to Fig  There is no question that the paper is timely and of broad interest, yet given that in vivo experiments shown have several significant caveats, and in vitro experiments largely replicate known results (Martin et al. 2006 andWillcockson et al., 2021), I am not fully convinced the paper is ready for publication until the authors are able to disentangle defect in K27me3 spreading from overall decrease in K27me3 observed in their system at steady state.
Reviewer #3 (Remarks to the Author): In the manuscript "Histone H1 facilitates restoration of H3K27me3 during DNA replication by chromatin compaction", Liu et al. reported that H1 facilitates the rapid post-replication restoration of H3K27me3 on repressed genes using ChOR-seq. The authors also showed that H1 facilitates the propagation of H3K27me3 by PRC2 in vitro and that the H3K27me3 restoration on nascent DNA is compromised in H1-TKO mESCs. Overall, the experiments were well performed, and results were clearly presented. ChOR-seq provided specifics on the rapid restoration of H3K27me3 following DNA replication, although H1 facilitating H3K27me3 propagation is somewhat expected given the substrate preference of PRC2 for H1 containing nucleosomes (Martin C 2006). The drastic effects by H1-TKO in mESCs observed in this study (dramatic gene expression changes (>1200 genes) and H3K27me3 ChIPseq), however, are in stark contrast to findings from previous studies. It has been well documented that H1-TKO produces limited expression changes /negligible changes in H3K27me3 ChIPseq in undifferentiated mESCs but induces dramatic changes in more specialized/differentiated cells (Fan 2005, Zhang 2012, Geeven 2015, Encode, Willcockson 2021, Yusufova 2021. How many mESC lines were analyzed and how were these H1-TKO mESCs by CRISPR/Cas9 characterized? It's not clear why the authors used H1-TKO mESCs by CRISPR/Cas9 when only found H1e enrichment with H3K27me3 and in cluster A in mESCs. The significance of H1e in rapid restoration of H3K27me3 in vivo remains to be addressed. In this manuscript, Liu and co-workers aim to address the role of histone H1 in restoration of K27me3 levels after replication. This is an important question and the ChORseq method is the appropriate tool to address this. The authors use ChORseq with spike-in correction to analyse restoration kinetics and conclude that repressed regions with high H3K27me3 levels restore most rapidly (consistent with findings in HeLa cells, Reveron-Gomez et al., 2018). They conclude that the restoration rate of H3K27me3 is pivotal for gene silencing, but no causative data are presented (repressed genes restore most rapidly, this does not mean that rapid restoration is required for silencing). The authors go on to show a positive correlation of the restoration kinetics to H1 occupancy, which reflects that there is more H1 at high occupancy K27me3 sites (known to restore most rapidly). The authors take advantage of H1-TKO to address the role of H1 in I overall find this story compelling, mainly because the model is highly consistent with earlier studies and most probably correct. It is well documented by the Skoultchi and Zhu labs that PRC2 activity and H3K27me3 occupancy is dependent on compaction and histone H1 (conclusions of Figure 5 and Figure 3). The novelty of this manuscript would be to demonstrate the relevance of this in restoration after replication. However, the conclusions on H3K27me3 restoration are not supported by the data as there are serious issues with the ChORseq experimental setup and data analysis. Also, a number of conclusions on causality are based on correlative observations. These serious concerns must be addressed before publication of the manuscript can be recommended.
Major points 1.The authors perform ChORseq in a synchronization set-up where mESCs are labeled after release into S phase 2hrs. There is no analysis of EdU labelled DNA and the genomic coverage, thus it is unclear what part of the genome they are tracking. If the aim is to address H3K27me3 dynamics across the genome and identify key parameters for restoration kinetics, the full genome must be covered by EdU labelling. The authors therefore would need to repeat the ChOR-seq experiments by labelling asynchronous cells.

Response:
We thank reviewer for this suggestion. Accumulating studies have showed that heterochromatin regions are replicated at middle and late S phase, but euchromatin regions prone to be replicated at early S phase. In addition, according to Anja Groth lab`s results, EdU labeled the vast majority of H3K27me3 peaks (~60-70%) in Hela S3 cells at middle S phase (Nazaret Reverón-Gómez, et al. Mol Cell, 2018). In our study, we found that the S phase lasts ~4 hrs for mESCs, and cells mainly entered middle S phase after 2 hrs (T0) of releasing (Response1. Fig.1A). These results suggest that this time point is suitable for labeling H3K27me3 regions in mESCs. As expected, genome coverage analysis showed that EdU incorporated into ~70% of H3K27me3 peak regions within 15 mins in the synchronized experiments (Response1. Fig.1B). In order to improve labeling coverage, we performed experiments in asynchronous mESCs with extended EdU-labeling time (20 mins), genome coverage analysis showed that ~80% of H3K27me3 peak regions are labeled by EdU in this condition, which largely overlapped with H3K27me3 peak regions labeled in synchronized mESCs (Response1. Fig.1B and 1C). In addition, ChOR-seq revealed that H3K27me3 exhibited similar restoration kinetics on the nascent chromatins as revealed in synchronized mESCs (Response1. Fig.1D, also in main manuscript Fig.S1G). However, the time window suitable for mapping H3K27me3 restoration is different in synchronized and asynchronous mESCs (0 2 4 6 hrs VS 0 1 2 3 hrs after EdU labeling) (in manuscript Fig.1B and Fig.S1G). In ChOR-seq, to prevent cell entering next S phase, we blocked mESCs with thymidine 3 hrs after EdU labeling (T3) in synchronized experiments, which endow us tracing the restoration of H3K27me3 during one cell cycle. However, in asynchronous condition we found that a portion of late S phase cells rapidly pass through G2/M and next G1 phase, and enter next S phase after 3 hrs of EdU labeling.
Addition of thymidine before T3 would cause S phase arrest for mESCs that are in early S phase when we label them with EdU. Hence, thymidine blockage at T3 time point in asynchronous mESCs is not efficient as that in synchronized mESCs. Consistent with this, ChOR-seq showed a reduction of H3K27me3 signals at its peak regions after T3 (Response1. Fig.1E). For these reasons, we represented and analyzed asynchronous ChOR-seq (T0-T3) in the manuscript and got similar results. Anyhow, these results suggest that synchronized and asynchronous conditions are both suitable for studying epigenetic restoration post-replication, but each condition has its own pros and cons. 2. There is little information provided about the data analysis, but it appears that the ChORseq analysis is done without filtering for replicated regions, which is essential in a synchronization set up. By including regions that are not covered by the EdU labelling, conclusions will be based on changes in background signal (i.e, regions that have not been replicated in a high proportion of the cells or not replicated at all in the 15min EdU pulse during early/mid S).

Response:
We thank the reviewer for raising this point. In the revised manuscript, we have filtered EdU-labeled H3K27me3 regions from whole H3K27me3-enriched regions, and revealed that EdU labels almost 70% and 80% of H3K27me3 peak regions in synchronized and asynchronous mESCs, respectively (Response1. Fig. 1B). In this case, we reanalyzed the restoration kinetics of H3K27me3 at EdU-labeled H3K27me3 regions and draw the same conclusion as previously gotten. We have modified figures 3.The use of a synchronization setup is also problematic for the comparison between WT and H1 TKO cells, as it assumes that the same set of regions will be replicating during the EdU pulse in early/mid S in both cell lines. This problem would be circumvented by doing ChORseq in asynchronous cells.

Response:
We agree with reviewer that it would be a problem to investigate the role of H1-compacted chromatin on H3K27me3 restoration if H1-TKO significantly alters the timing of replication. So, we analyzed the genome coverage of EdU in wild-type and H1-TKO mESCs and found that a large set of genome regions are simultaneously labeled by EdU in synchronized wild type (~69.84% of all labeling regions) and H1-TKO (~70.62% of all labeling regions) mESCs (Response1. Fig.2A left). However, under asynchronous condition, we found that ~52.78% of EdU-labeled genome regions in wild type mESCs are overlapped with 45.88% of EdU-labeled genome regions in H1-TKO mESCs (Response1. Fig.2A S4D). These results suggest that H1-TKO indeed partially alters replication timing, while the replication domain used in H3K27me3 peak regions are preserved in wild type and H1-TKO mESCs at a large extent. In addition, we also performed ChOR-seq in HA-AID-H1c/e inducible degradation system, which largely circumvents this concern (Response1. H3K27me3 restoration kinetics was also previously shown to be fastest at sites with high K27me3 and PRC2 binding (Reveron-Gomez et al., 2018). It would be advisable to cite these publications when concluding on experiments that validate or corroborate previously findings (even if the papers are cited elsewhere in the manuscript). Given the K27me3 level is 2-fold reduced in the H1 TKO cells, it not clear whether the lower K27me3 or lack of H1 is responsible for the reduced restoration kinetics. An inducible system with similar baseline prior to replication would be preferable. The authors could also compare in WT cells restoration kinetics of regions with similar K27me3 levels and different H1 density. There are conclusions on causality based on correlations mainly concerning the importance of restoration rates for gene regulation.

Response:
We agree with reviewer for this concern. To elucidate whether the reduced H3K27me3 level would affect the restoration of H3K27me3 in H1-TKO mESCs, we generated an inducible H1c/e degradation system, in which the HA-AID was knocked into the N-terminus of H1c and H1e genes simultaneously using CRISPR/Cas9 mediated-gene editing in 9×MYC-TIR1 stable expression mESCs (HA-AID-H1c/e cell line). In this system, the HA-AID-H1c/e proteins are gradually degraded through ubiquitin-proteasome system when IAA (indole-3-acetic acid) was added into the culture medium (Response1. Fig.3A and 3B). We found that the synthesis and degradation of HA-AID-H1c/e reach equivalent at ~12 hrs point after IAA treatment, however the H3K27me3 level don`t significantly changed before ~24 hrs, endowing us at least ~12 hrs to investigate the restoration kinetics with an equal H3K27me3 initial level. In addition, the results are also consistent with the original findings that reduction of H3K27me3 mainly caused by passive dilution during cell cycle other than turnover and demethylation (Nazaret Reverón-Gómez, et al. Mol Cell, 2018). Basing on these findings, we first cultured HA-AID-H1c/e cell line in IAA containing medium 6 hrs then equally divided the cells into 2 sets (Response1. Frankly, comparing the restoration kinetics of regions with similar H3K27me3 levels and different H1 density is a good idea to elucidate the role of H1-compacted chromatin in regulating the restoration of H3K27me3 post-replication. However, we unfortunately can`t collect a set of these regions to perform statistical analysis, because we found that the levels of H3K27me3 are broadly positive correlated with the levels of H1 proteins on chromatin genome-wide (Response1. Fig.4, also in manuscript Fig.3A and 3B).

Response:
We thank reviewer for this suggestion. We have provided the information in Data Accession of revised manuscript and in "reporting summary" accompanying with this manuscript.
2.The authors refer to different genomic regions without defining them or providing any information on their overlap, e.g. they classify first restoration kinetics of peaks and then calculate the restoration kinetics to pre-defined promoters (ES, housekeeping, bivalent etc.). It is also not clear if these promoters have H3K27me3.

Response:
We are sorry for the confusion. Indeed, in our study all genes that we analyzed are H3K27me3 targets. To achieve this, we first collected the gene list of various categories (house-keeping, es-specific, bivalent and tissue-specific) from previous publications, and then filtered EdU-labeled H3K27me3 target genes from individual gene list. By this means, we determined the four categories of H3K27me3 target genes for further analysis. It is worth noting that the number of bivalent genes accounts for the majority of H3K27me3 target genes (Response1. Chart 1). We have added the details to the revised manuscript (page 6). 3.Details missing in the figures and figure legends, mostly units and data source. Details missing in the methods, e.g there is a second thymidine addition in the schematic in

Response:
We thank reviewer for this suggestion. We have revised the manuscript thoroughly and added the missing details in Figure 1A, figure legends (1A, 1E, 2D-2G, 2J, 4I) and Methods sections (page 17) that were highlighted in the manuscript. We also added data source in Data Accession section.

Reviewer #2 (Response to the reviewer):
Mechanisms of propagation and maintenance of epigenetic regulation across cell cycle are of significant interest, both because of fundamental importance to developmental biology, and due to emerging understanding that many human disorders are driven by misregulation of gene expression. The idea that repressive chromatin modifications are particularly dependent on the "inheritance" mechanisms during S phase has been   Fig.1B). It argues that heterochromatin is replicated later than active chromatin. Besides, our ChOR-Seq and enrichment analysis showed that a portion of H3K27me3-highly-enriched peaks (28.73%) are restored rapidly during T0-T2 (main manuscript Fig.1B and Fig.S1F), demonstrating H3K27me3 is rapidly restored at dense heterochromatin regions following DNA replication (main manuscript Fig.1E). In addition, T0-T2 is the first 2 hrs of chasing (2.15-4.15 hrs after release). Response2. Figure1 their dynamics (change from T0 to T6) and original peak intensity (top to bottom in "Parental" heatmap. Given that much of the paper is based on interpretation of these data, it's critical to show that there are indeed distinct restoration clusters. Figure 1B looks like it, and perhaps some of the discrepancy is due to the scale differences (S1B is scaled -3 to 3, and 1B is scaled -1 to 1). Yet I do not see how clusters A, B and C are derived from raw data shown in S1B.

Response:
We thank reviewer for this suggestion. Accordingly, we have shown the clustered H3K27me3 signals by a new Heat-map in revised manuscript (in manuscript I am not convinced that this system allows the authors to unequivocally state that H1 loss is directly affecting the rate of spreading. We know that H1 appears to stimulate PRC2 even in di-nucleosomal substrates (first reported by Martin et al., JBC 2006) -I think the jump to compaction as mechanism is intriguing and very plausible, but is not fully supported by data. Further, comparing data in 2C-G, I am left with the impression that the greatest predictor of K27me3 restoration rate (Fig. 2C) is K27me3 read density ( Fig 2D) -not the H1e read density (Fig 2E).

Response:
We agree with reviewer for this concern. To circumvent this problem, we generated a H1c/e inducible degradation system (HA-AID-H1c/e cell lines). In this system, the HA-AID-H1c/e proteins are gradually degraded through ubiquitinproteasome system after adding IAA (indole-3-acetic acid), but bulk H3K27me3 do not significant changed during the tracking period of H3K27me3 restoration. Then we performed ChOR-seq and found that H1c/e reduction resulted a significant delay of H3K27me3 restoration after DNA replication (Response2. Fig.3A-H, revised manuscript Fig.5), which supports the conclusion that H1-mediated chromatin compaction have an important role in promoting H3K27me3 restoration after DNA replication. Response2. Figure3 In addition, using in vitro HMT assays we found that PRC2 and H3K27me3 indeed have a positive feedback loop (consistent with previous findings), and H1 can further enhance PRC2 HMT activity on poly-nucleosomes through compacting chromatin fibers (Response2. Fig.3I and J, revised manuscript Fig.6 and Fig. S6), suggesting that "positive feedback loop" and H1-compacted chromatin are two independent mechanisms, and they could cooperatively promote the robust spreading of H3K27me3 and the fast restoration of H3K27me3 post-replication in cells. We have shown the data related to H1c/e inducible degradation system in " Fig.5" in revised manuscript. We also described this finding in the "response letter" to reviewer 1.  et al. Nature, 2021;Geert Geeven, et al. Genome Biol, 2015). Hence, the alteration of mRNA in H1-TKO mESCs should be a superimposition effect of these mechanisms mentioned above. However, in fact there are studies showing H1 knockout mainly cause up-regulation of genes in somatic cells, such as lymphocytes (Nevin Yusufova, et al. Nature, 2021), suggesting that H1s differentially regulate gene expression among distinct type of cells.
In our revised manuscript, we filtered mis-regulated H3K27me3 targeting genes (299) and found that 56.19% (168) were down-regulated and 43.81% (131) were derepressed among mis-regulated H3K27me3 targeting genes. These results indicated that H1 also regulates other transcriptional regulation pathways at H3K27me3-enriched regions such as DNA methylation, H3K9me and HP1 and so forth, and it seems that the pathway we identified maybe dominantly involved in regulation of these up-regulated genes (131) compared with other mechanisms, at least, in mESCs. Distribution analysis showed that cluster A covered a large set of these mis-regulated including up-and down regulated genes (Response2. Fig.4), indicating that the chromatin states in cluster A should experience a dramatical change following H1c/d/e knockout. We have revised the manuscript in page 8.
In addition, we deleted the GSEA plots in the revised manuscript, because we agree that there is no conception novel for the present study. Response2. Figure4 5. The previous concern could be partially alleviated by a more robust distinction between recruitment and spreading. If H1 is indeed affecting the spreading dynamics, then the K27me3 peak height should remain roughly similar -but the breadth is expected to be reduced dramatically. At the very least, I'd expect a faster dropoff (such that K27me3 peaks in WT would essentially represent "obtuse angles", and in TKO would be more akin to "acute angles") from the peak center to the edges. The authors do the right experiment in Fig 5E-F, but looking at the data, I don't see a difference in K27me3 peak shape -only an overall decrease in H1 TKO background -therefore, I still can't parse out whether H1 loss causes defect in "recruitment" or "spreading" in vivo.

Response:
In targeting experiment (Fig 5E-5F), the shape of H3K27me3 peak is largely dependent on the primer sets used in ChIP-qPCR, which may alter when choosing other sets of primers. To rule out this possibility, we repeated the targeting experiments and quantitated H3K27me3 enrichment by ChIP-seq. In addition, the HMT and spreading activation of PRC2 requires heterodimer of EZH2 and EED subunits, so we this time co-overexpressed HA-EZH2-TetR/FLAG-EED or HA-EED-TetR/FLAG-EZH2 in 8×TetO targeting cells, which could additionally augment the difference of H3K27me3 spreading between wild type and H1-TKO mESCs compared with single HA-EZH2-TetR overexpression. Our ChIP-seq results showed that targeting HA-PRC2-TetR efficiently deposits H3K27me3 across 8×TetO sites with width distribution in wild type cells, and the peak height of H3K27me3 is 10× lower and the width is narrowed in H1-TKO (vertical ordinate for H1-TKO is zoomed in 3 times in the picture show) (Response2. Fig.5D). In our previous study (Jicheng Zhao et al, NCB, 2020, Fig.3j), we have tested the effect of H1 loss on the "recruitment/targeting" of HA-TetR-proteins and found that the height of HA-TetR-RING1B peaks in H1-TKO is the same as that in wild type mESCs at 8xTetO peak center (Response2. Fig.5A), suggesting that H1 loss don`t significantly intervene the binding of HA-TetR-proteins with TetO arrays. In addition, we found that H1 didn`t affect PRC2 activity on mono-nucleosome (manuscript Fig.S6E, Response2. Fig.5B), demonstrating that H1 don`t intervene the interaction between enzymes and nucleosomes. Besides, it is widely accepted the notion that open chromatin would be more accessible for enzymes than H1-compacted chromatin, so PRC2 should inert on H1-condensed chromatin if no other mechanism exists, but we in fact observed the opposite results (PRC2 is more active on H1condensed chromatin) (Response2. Fig.5B and 5C; also showed in main manuscript Fig.6B and Fig.S6E). Therefore, these results altogether suggest that H1-compacted chromatin has an important role in H3K27me3 restoration by promoting the spreading of H3K27me3. According with our results, it seems that the recruitment, both positive  Fig.6B top). After staining, we respectively estimated the "gray valve" of linker histone H1s and core histone for each lane using ImageJ (subtracting the background). Then we calculated the ratio of H1/core histones for wild type and H1-TKO at the series of loading condition. Next, the reduction of H1s in H1-TKO mESCs relative wild type mESCs were estimated by the formula showed in Response2. Fig.6B  Interestingly, the results showed that the H3K27me3 is relatively homogenous in these cells (Response2. Fig.7A-7C), which rules out the possibility raised by review. In addition, we believe this concern can also be resolved by the H1 inducible degradation experiments. As mentioned, we also reanalyzed the studies carried out by Skoultchi lab and confirmed that they also found ~2 fold reduction of H3K27me3 in H1-TKO mESCs (Response2. Fig.8). Besides, in the previous studies by Skoultchi lab they did the immunofluorescence for H3K27me3, but didn't show the results in their paper.
c. To add to Fig S4A, I'd like to see more detailed quantification of cell cycle progression in these cells. E.g., it appears that T4 plots are a bit different -is it an outlier or part of the phenotype?
Response: Thank reviewer for pointing this issue out. We reanalyzed the biological repeated experiments data for cell cycle analysis and confirmed that the cell cycle of wild type and H1-TKO are roughly the same (Response2. Fig.9). The cell cycle difference between wild type and H1-TKO at T4 is a deviation in each batch of FACS.
Basing on our experiences, the cell number and time spent on ice during staining before FACs all slightly contributes to the signal deviation. In the revised manuscript, we replaced old data by biological repeated data and we also added the vertical ordinate in Coomassie gels shown in Fig. 5 should be cropped above 30 kDa size to account for H1.
Response: Thank reviewer for pointing this issue out. In our study, in order to generate enough highly purified recombinant H1e proteins we have optimized the process for H1 purification. Briefly, we first harshly washed bacteria inclusion bodies, which contain recombinant H1e proteins, to remove contaminants as possible as we can.
Second, we resolved H1e from bacteria inclusion using 1M NaCl, then sequentially purified with Heparin column and hydroxyapatite column as we described in the methods section. Finally, we identified the purity and stability of H1e using SDS-PAGE and Coomassie staining, and we found that the recombinant H1e purified using this protocol is pure and relative stable (vast majority of the H1e is full length) even after a long period of storage at -80℃ (Response2. Fig.10). We have shown the Coomassie staining result of H1e in the revised manuscript ( Figure 6 and Figure S6). Response2. Figure10 spreading from overall decrease in K27me3 observed in their system at steady state.
Response: Thank reviewer for these suggestions. Accordingly, we have added all necessary information in the revised manuscript ( Figures 1B, 1E, S1G and S4E; Figure   legend of Figures S1E, Figures 3A and 3D).